Method of inducing antibody production against an infectious agent in a host

ABSTRACT

The present invention relates to synthetic herpes simplex virus (HSV) promoters which are constructed by operatively linking the 5&#39; nontranscribed domain of an HSV α gene to a fragment containing the transcription initiation site and the 5&#39; transcribed noncoding region from an HSV γ gene. Synthetic promoters of the invention that are operatively linked to heterologous genes, inserted into HSV genomes and used to generate live virus are useful for expressing polypeptides encoded by the heterologous genes in appropriate host cells. The synthetic promoters direct transcription of the heterologous genes constitutively throughout the reproductive cycle of the virus at a high cumulative level. The recombinant viruses of the invention can also be used as vaccines to present polypeptides against which a host will mount an immune response.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. USPHSR35 CA47451 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/332,467 filed Oct. 31, 1994, now U.S. Pat. No. 5,641,651, which is acontinuation of U.S. patent application Ser. No. 07/996,961 filed Dec.23, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to synthetic herpes simplexvirus (HSV) promoters useful for expressing heterologous genes withinthe context of the HSV genome. More specifically, the invention relatesto a synthetic viral promoter including the 5' nontranscribed domain ofan HSV α gene operatively linked to the transcription initiation siteand 5' transcribed non-coding domain of an HSV γ gene.

BACKGROUND

The HSV-1 genome includes at least seventy-six open reading frames whichencode at least seventy-three diverse polypeptides. The genes encoded bythe viral genome have been classified into three major groups designatedas α, β and γ, whose expression is coordinately regulated andsequentially ordered in a cascade fashion. The α genes are expressed inthe absence of prior viral protein synthesis. Expression of the α genesis induced by the interaction of an HSV structural protein designated asα-transinducing factor or virion protein No. 16 and several hostproteins which interact with a response element located in the 5'untranscribed domains of all α genes. The products of the α genestrans-activate the expression of the β genes by a mechanism as yetunknown, but the response elements for the induction of the β genesappear to reside in their 5' untranscribed domains. Among the responseelements commonly associated with β gene promoters are TATAA boxes, SP1binding sites, CCAAT boxes, and sites for binding of the α4 geneproduct. The expression of γ genes also requires the expression of αgenes. In addition, their expression is partially (the γ₁ genes) ortotally (the γ₂ genes) dependent on viral DNA synthesis. In contrast tothe α and β genes, in γ genes promoter response elements (including α4protein binding sites) have been reported to be present in both 5'untranscribed and transcribed non-coding domains.

Various HSV promoters have been utilized to express non-HSV genes in thecontext of the HSV genome. Post et al., Mol. Cell. Biol., 2:233-240(1982) reports the expression of ovalbumin from a fusion of a chickenovalbumin gene to the promoter of the α4 gene. Similarly, Hummel et al.,Virology, 148:337-343 (1986) describes the expression of an Epstein-Barrvirus protein, the EBNA1 protein, from the α4 promoter in the HSVgenome. Synthetic genes consisting of the coding sequences of a foreigngene fused to a specific HSV promoter and inserted into the HSV genomeare expressed in the temporal class of the promoter as is reported, forexample, in Shih et al., Proc. Natl. Acad. Sci. USA, 81:5867-5870 (1984)where the hepatitis B virus S gene specifying the hepatitis B virussurface antigen was inserted into the HSV genome under the control ofthe HSV α4 gene promoter and separately under the control of the HSVthymidine kinase gene β promoter.

While the HSV genome has been successfully utilized to express productsof heterologous genes, inserting foreign genes under the control ofnative HSV promoters is problematic because of the sequential order ofactivation of HSV promoters. A foreign gene under the control of an HSVα promoter is transcribed early in the replicative process of the viruswhile a foreign gene under the control of an HSV γ promoter istranscribed only in the later stages of replication. Heterologouspolypeptides or proteins are therefore not continuously expressedthroughout the infection cycle of the virus and the potential level offoreign protein synthesized is correspondingly reduced.

There thus continues to be a need in the art for HSV synthetic promotersspecifically designed to express gene products throughout the infectiousprocess of the virus and overproduce the gene product.

SUMMARY OF THE INVENTION

The present invention provides synthetic HSV promoters that include anHSV (i.e., HSV-1 or HSV-2) α gene promoter fragment operatively linked5' to an HSV (i.e., HSV-1 or HSV-2) γ gene promoter fragment. Alsoprovided are DNA constructs including a synthetic HSV promoter of theinvention operatively linked 5' to a heterologous gene, recombinant HSVgenomes containing such DNA constructs, and recombinant viruses havingsuch a recombinant HSV genomes.

The HSV α gene promoter fragment of the synthetic promoters of theinvention consists essentially of the 5' nontranscribed domain of an αgene. The α gene promoter fragment preferably includes the promotersequences upstream of the translation initiation site (i.e., cap site)and preferably is a fragment of the α4 gene. More preferably, the α4gene promoter fragment consists essentially of nucleotides -588 to -12of the α4 gene promoter relative to its transcription initiation site orof a functionally equivalent nucleotide sequence.

The HSV γ gene promoter fragment of the synthetic promoters of theinvention consists essentially of the transcription initiation site and5' transcribed non-coding domain of a γ gene. The γ gene fragmentpreferably includes the transcription initiation site and promotersequences downstream of the transcription initiation site and preferablyis a fragment of the γ₁ U_(L) 19 gene. More preferably, the γ₁ U_(L) 19gene promoter fragment consists essentially of nucleotides -11 to +189of the γ₁ U_(L) 19 gene promoter relative to its transcriptioninitiation site or of a functionally equivalent nucleotide sequence.

Specifically illustrating synthetic HSV promoters of the invention isthe synthetic promoter designated the α4-γ₁ U_(L) 19 promoter which wasdeposited in the plasmid pRB4297 in E. coli strain XL1 Blue with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 on Sep. 4, 1992 and was assigned ATCC Accession No.69068.

The synthetic promoter/heterologous gene DNA constructs of the inventionwhen included in recombinant HSV genomes and/or recombinant viruses areuseful for expressing heterologous polypeptides in appropriatelytransfected or infected host cells. (See Roizman, European Patent No.176,170 granted on Aug. 19, 1992 which is directed to the use of herpessimplex virus as a vector.) The heterologous genes may encode anynon-HSV polypeptide or protein of interest that confers immunity toinfection, for example, surface glycoproteins of Epstein-Barr virus,influenza virus, human immunodeficiency virus, papilloma viruses,varicella-zoster virus, human cytomegalovirus and human herpesvirusHHV-6, as well as HSV proteins not naturally under the transcriptionalcontrol of either of the component promoters of the syntheticconstructs. Transcription in a host cell from a synthetic promoter ofthe invention occurs constitutively throughout HSV infection and resultsin overproduction of the gene product. Specifically illustratingrecombinant herpes simplex viruses of the invention is the recombinantherpes simplex virus R7125 which was deposited with the ATCC on Nov. 19,1992 and was assigned ATCC Accession No. VR 2389.

The recombinant viruses described herein that include a heterologousgene operatively linked to a synthetic promoter of the invention areuseful as vaccines. A host (especially a human host) may be immunizedagainst a polypeptide of a disease-causing organism by administering tothe host an immunity-inducing amount of a recombinant virus of theinvention which produces the polypeptide. Immunization againstpolypeptides encoded by various non-HSV viruses and bacteria iscontemplated, including Epstein-Barr virus (EBV), varicella-zoster virusand cytomegalovirus, as well as immunization against various other humanherpesviruses. Herpes simplex viruses that are appropriate for insertionof the DNA constructs of the invention and for use as vaccines areavirulent strains (e.g., the strains described in Roizman, U.S. Pat. No.4,859,587 issued on Aug. 22, 1989 and in Roizman, PCT InternationalPublication No. WO 92/04050 published on Mar. 19, 1992).

Immunization of a human host with a recombinant herpes simplex virus ofthe invention involves administration by inoculation of animmunity-inducing dose of the virus by the parenteral route, preferablyby intramuscular or subcutaneous injection. Inoculation may also byeffected by surface scarification or by inoculation into a body cavity.Typically, one or several inoculations of between about 1000 and about10,000,000 plaque-forming units each, as measured in susceptible humanor nonhuman primate cell lines, are sufficient to effect immunization ofa human host. Virus to be used as a vaccine may be utilized in liquid orfreeze-dried form (in combination with one or more suitablepreservatives and/or protective agents to protect the virus during thefreeze-drying process).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the HSV-1 genome and the naturallocations of the promoters used in the construction of an α₄ -γ₁ U_(L)19 promoter of the invention.

FIG. 2 is a schematic representation of the structure of three syntheticHSV promoter DNA constructs respectively included in recombinant virusesR3213, R7610 and R7600.

FIG. 3 is an autoradiographic image of chicken ovalbuminimmunoprecipitated from the extracellular medium of cells respectivelyinfected with HSV-1(F) (wild type HSV) and recombinant viruses R3213,R7610 and R7600.

FIG. 4 is a graph depicting the relative amounts of chicken ovalbuminmade in cells during infection with HSV-1(F) and recombinant virusesR3213, R7610 and R7600.

FIG. 5 is a schematic representation of the genomes of HSV-1(F) and ofrecombinant viruses R3410, R7020 and R7125.

FIG. 6 is a schematic representation of the synthetic HSVpromoter/Epstein Barr virus glycoprotein gp350/220 DNA construct inrecombinant virus R7125.

FIGS. 7A and 7B are autoradiographic images of HSV-1(F), recombinantvirus R7020, nonpassaged recombinant virus R7125 and serially passagedR7125P7 DNA digested with EcoRI or EcoRV, respectively, and hybridizedto DNA encoding Epstein Barr virus glycoprotein gp350/220.

FIG. 8 is an autoradiographic image of gp350/220 immunoprecipitated fromcells infected with recombinant virus R7020 and with recombinant virusR7125.

DETAILED DESCRIPTION

The following detailed description illustrates practice of the inventionin the context of a single illustrative synthetic promoter whose designis based on the observation that the response elements fortransactivators of HSV α genes are located upstream of the transcriptioninitiation site whereas γ gene response elements are found downstream ofthe transcription initiation site. Construction of the syntheticpromoter thus takes advantage of the hitherto unrecognized prospect thatthe 5' untranscribed domain of an α gene, when fused to the 5'transcribed non-coding domain of a γ gene, forms a promoter thatexpresses a gene product throughout HSV infection and overproduces thegene product.

The HSV-1 α promoter fragment selected for illustration here consists ofthe nucleotides -588 to -12 of the α4 promoter relative to itstranscription initiation site. This sequence includes several α-TIFresponse elements, several SP1 binding sites, and the native α4 TATAAbox. For the γ promoter fragment, a domain of the γ₁ U_(L) 19 geneconsisting of nucleotides -11 to +189 relative to its transcriptioninitiation site was selected. The U_(L) 19 gene encodes virion proteinVP5, the major capsid protein of the virus. Previous studies see Honesset al., J. Virol., 14:8-19 (1974); Honess et al., Proc. Natl., Acad.Sci. USA, 72:1276-1280 (1975); and Roizman et al., Eds., TheHerpesviruses, Vol. 3, Plenum Press, New York, 45-104 (1984)! reportthat VP5 is an abundant protein whose synthesis is only partiallyaffected by inhibitors of DNA synthesis.

Numerous aspects and advantages of the present invention will beapparent upon consideration of the following examples relating to theconstruction and use of synthetic herpes virus promoters. Moreparticularly, Example 1 describes the construction of the syntheticα4-γ₁ U_(L) 19 promoter, fusion of the synthetic promoter to a chickenovalbumin gene in a recombinant HSV-1 genome, and generation of arecombinant virus, designated R7600, therefrom. Example 2 describes thetesting of recombinant virus R7600 for the capacity to produce ovalbuminand an analysis of the production of ovalbumin by the recombinant virusduring infection of host cells in comparison to control viruses. Example3 describes the construction of a DNA construct including the syntheticα4-γ₁ U_(L) 19 promoter fused to structural sequences encoding theEpstein Barr virus glycoprotein gp350/220 and the generation of arecombinant virus, desginated R7125, having the DNA construct within itsgenome. Example 4 describes the testing of recombinant virus R7125 forthe capacity to produce gp350/220 in comparison to a control virus andthe stability of the recombinant virus after serial passage.

EXAMPLE 1

FIG. 1 schematically depicts the HSV-1 genome and the locations ofrestriction fragments of the genome which contain the promoter fragmentsof the HSV-1 U_(L) 19, U_(L) 44 (or gC) and α4 genes utilized herein. InFIG. 1, ab and b'a' represent the inverted repeats flanking the U_(L)components, whereas a'c' and ca represent the inverted repeats flankingthe U_(s) component. The locations of BamHI fragments B', I and N whichrespectively contain the U_(L) 19, gC and of α4 promoters are indicated.

A synthetic HSV promoter, designated the α4-γ₁ U_(L) 19 promoter, wasconstructed as follows. DNA of bacterial plasmid clone pRB140 Post etal., Proc. Natl. Acad. Sci. USA, 77:4201-4205 (1980)!, which containsthe BamHI B' fragment of the wild type HSV-1(F) strain (ATCC VR 733)including portions of the promoter of the U_(L) 19 gene, was subjectedto digestion with restriction enzymes BamHI and SalI. The resulting˜1074 base pair (bp) fragment was ligated into the vector pGEM3z(Promega, Madison, Wis.) which had been digested with BamHI and SalI toyield pRB4291. DNA from pRB4291 was then digested with BamHI and BstYI,and the 200 bp fragment which resulted was ligated into pGEM3z digestedwith BamHI to create pRB4294. This clone contains five tandem copies ofthe fragment representing the region -11 to +188 of the U_(L) 19promoter relative to the transcriptional start site of the U_(L) 19 RNA.

A second plasmid, pRB4295, containing promoter elements of the α4 genewas derived from pRB168 (Post et al., supra) which contains the BamHIfragment Z of HSV-1(F). Plasmid pRB168 DNA was digested with NarI andBglI and a 420 bp fragment was isolated and ligated to a syntheticdouble stranded oligonucleotide (the sequence of which is set out below)which conforms in part to elements from -88 to -12 in the α4 promoterregion (see description of pRB4295 below for differences). ##STR1## Theligation product was redigested with NarI to uncouple fragments whichhad been ligated in tandem at the -520 NarI site. After repurification,the appropriate 500 bp product was ligated into pGEM3z which had beendigested with AccI and BamHI to yield pRB4295. pRB4295 contains thenatural α4 promoter elements from -520 to -88, natural sequence restoredby the oligonucleotide from -88 to -40, then two nucleotide base changesto introduce a NotI recognition site (underlined). Natural sequences arerestored from -37 through the natural TATA element at -28 (bold) to aninduced BamHI recognition site at -12 (double underlined).

A 200 bp fragment containing a portion of the 5' untranscribed sequence,the transcription initiation site and the 5' transcribed non-codingsequence of the γ₁ U_(L) 19 gene (i.e., the transcription initiationsite and sequences downstream of the transcription inflation site of theγ₁ U_(L) 19 gene) was excised from pRB4294 with BamHI and inserted intothe induced BamHI site of pRB4295, such that base -11 of the γ₁ U_(L) 19fragment was adjacent to base -12 of the α4 promoter to generate theplasmid designated pRB4297 (ATCC 69068). The DNA sequence of thesynthetic α4-γ₁ U_(L) 19 promoter is set out in SEQ ID NO: 2, whereinnucleotides 1 to 594 correspond to the α4 gene promoter fragment,nucleotides 595 to 797 correspond to the γ₁ U_(L) 19 gene promoterfragment, and nucleotide 606 is the transcription start site.

Plasmid pRB4297 was digested with KpnI and HindIII and the resulting 700base pair fragment containing the α4-γ₁ U_(L) 19 synthetic promoter wasinserted into the KpnI site of pRB4302 to generate plasmid pRB4303.pRB4302 contains an entire HSV-1 genome with a cDNA copy of thestructural sequences of the chicken ovalbumin gene inserted into theBglII site in the leader of the thymidine kinase (tk) gene of the HSV-1Bam Q fragment (see FIG. 1). The KpnI site of pRB4302 is adjacent to thetranslational start site of the chicken ovalbumin gene. The orientationin which the synthetic α4-γ₁ U_(L) 19 promoter was inserted into pRB4303was the correct transcriptional orientation with respect to thetranslation start site of the ovalbumin gene. A schematic representationof the synthetic HSV promoter/ovalbumin gene DNA construct in pRB4303 isset out in FIG. 2, wherein the wide box labeled "ova" represents thecDNA sequence of the structural portion of the chicken ovalbumin gene,the thin boxes represent the promoter regions and the arrowhead denotesthe transcriptional orientation of the promoter.

The synthetic HSV promoter/ovalbumin DNA construct was inserted into anHSV-1 genome by recombination through thymidine kinase sequencesflanking the DNA construct by a method similar to that described in Postet al., Cell, 25: 555-565 (1981). Specifically, cotransfection of rabbitskin cells with DNA from pRB4303 and with DNA from the wild type HSV-1strain HSV(F), followed by selection on 143TK cells for the TK⁺phenotype produced a number of recombinant viruses which then wereplaque purified twice on Vero cells. DNA was isolated from eachrecombinant virus, digested with KpnI, and checked by Southern blotanalysis for a change in the electrophoretic mobility of the Kpnfragment which would indicate the acquisition of pRB4303 sequencescontaining the synthetic HSV promoter/ovalbumin construct. Of thefourteen recombinant viruses tested, five contained the α4-γ₁ U_(L) 19synthetic promoter/chicken ovalbumin gene construct.

EXAMPLE 2

The five recombinant viruses generated in Example 1 were tested for thecapacity to produce chicken ovalbumin. By a method described inArsenakis et al., Methods in Molecular and Cellular Biology, 2:5-16(1990), all five were shown to produce comparable amounts of ovalbuminwhen the extracellular medium of infected cells was electrophoresed indenaturing polyacrylamide gels. One of these five viruses, designatedR7600, was chosen for temporal analysis of ovalbumin expression duringthe HSV infection cycle by detection of the protein excreted frominfected cells by immunoprecipitation with commercially availableanti-ovalbumin antibody.

HSV-1(F) and two different HSV-1 recombinant viruses were used ascontrols in the experiments. The first recombinant virus, R3213,contains the nucleotides -1467 to +33 of the α4 promoter fused to thechicken ovalbumin gene and inserted into the BglII site of the tk genein the HSV-1 genome as present in intermediate plasmid pRB3213. SeeArsenakis et al., supra, and FIG. 2. The second, R7610, containsnucleotides -2700 to +137 of the promoter of the HSV glycoprotein C gene(U_(L) 44) fused to the chicken ovalbumin gene and similarly insertedinto the HSV genome. See FIG. 2. Like the product of the U_(L) 19 gene,the product of the U_(L) 44 gene is made in abundant mounts. The R7600virus of the invention and the three control viruses were propagated onHEp-2 cells and titered on Vero cells.

Replicate cultures of HEp-2 cells were each infected with 10plaque-forming units (pfu) of HSV(F), R7600, R3213 or R7610 viruses. At2, 5, 8, 11, 14, and 17 hours post infection, the medium was replacedwith 1 ml of 199V (JRH Bioscience, Lenexa, Kans.) without calf serum,1/10 the normal concentration of methionine, and 100 μCi of ³⁵S-methionine (>1000 Ci/mmol; Amersham, Arlington Heights, Ill.). Onehour later the medium was collected, clarified by centrifugation, andconcentrated 10-fold. Chicken ovalbumin was then collected from eachsample by immunoprecipitation with anti-chicken ovalbumin antibody(Organon Teknika, West Chester, Pa.) and Protein A conjugated sepharosebeads (Sigma Chemical Co., St. Louis, Mo.). The ovalbumin was elutedfrom the beads with disruption buffer containing sodium dodecyl sulfate,electrophoretically separated on a 9.5% denaturing polyacrylamide gel,electrically transferred onto a nitrocellulose sheet (Scheicher andSchuell, Keene, N.H.) and visualized by its reactivity with antibody,autoradiography, or both. In some experiments the amount of labeled ³⁵S-methionine labeled ovalbumin was quantified with a Betascope 603(Betagen, Waltham, Mass.). An example of the results obtained by theseprocedures is an autoradiogram presented in FIG. 3. Similar results arepresented graphically in FIG. 4.

The timing and level of chicken ovalbumin expression in R7600-infectedcells during early infection were nearly identical to that observed inR3213-infected cells (because the ovalbumin gene is under the control ofan α promoter in the R3213 virus). At 9 hours post infection, whenproduction was decreasing in R3213-infected cells, the level fromR7600-infected cells stabilized. After 9 hours, the level of ovalbuminfrom R3213-infected cells declined until no ovalbumin was detectable,while the level from R7600-infected cells increased dramatically. Thisincrease occurred at approximately the time in HSV infection thatexpression switches from the α and β genes to the γ genes. The amount ofchicken ovalbumin produced by R7600-infected cells during the late phaseof the infection was much greater than that from R7610-infected cellseven though the ovalbumin gene was under the control of a γ promoter inthe R7610 virus.

Over the course of vital infection, the yield of chicken ovalbumin fromR7600-infected cells was at least 57-fold greater than the backgroundmeasured in the medium of cells infected with HSV(F), 5.2-fold more thanproduced by R7610-infected cells, and 3.8-fold more than in cellsinfected with R3213.

EXAMPLE 3

Epstein-Barr virus (EBV) infects and predominantly remains latent inhuman B lymphocytes. The virus is mainly associated with subclinicalinfection, but primary infection with EBV is the cause of infectiousmononucleosis. Recent interest has focused on EBV because it may play arole as a human carcinogenic agent in the etiology of Burkitt's lymphomaand nasopharyngeal carcinoma. Immunization against EBV is thus indicatedfor individuals susceptible to or infected with the virus.

The synthetic α4-γ₁ U_(L) 19 HSV promoter described in Example 1 wasfused to an EBV gene encoding the vital glycoprotein gp350/220. Arecombinant virus, designated R7125, having the synthetic HSVpromoter/gp350/220 synthetic gene as part of its genome was derived froma stable genetically engineered attenuated strain of HSV-1 designatedR7020 (ATCC VR2123). See Meignier et al., J. Infect. Dis., 158:602-614(1988)and Meignier et al., J. Infect. Dis., 162:313-322 (1990). See alsoFIG. 5 wherein line 1 represents the genotype of wild type HSV(F); line2 indicates the deletions present in R3410 (ATCC VR2124) to attenuatethe virus (R3410 has deletions in the tk, U_(L) 24, U_(L) 55, U_(L) 56genes and only has single copies of α4, α0, and γ34.5 genes and thelatency associated transcript sequence); line 3 depicts the addition ofthree glycoprotein genes from HSV-2(G) (ATCC VR734) and of an α4promoter driven tk gene (in a nonnatural site) to R3410 to create R7020(R7020 expresses gG-2, gD-2, gI-2 and αtk); and the fourth line showsthe introduction of the synthetically driven gp350/220 into the localeof the deleted site of tk and U_(L) 24 in R7125.

To link the EBV gp350/220 coding sequences to the α4-γ₁ U_(L) 19promoter, pRB4297 (the plasmid described in Example 1 which contains theα4-γ₁ U_(L) 19 synthetic promoter) was cut with KpnI and HindIII togenerate a fragment including the promoter. The sticky ends of thefragment were blunt-ended with T4 DNA polymerase and deoxynucleotides,and the fragment was ligated to the DNA of clone pMA102 (containing theBLLF-1 gene of EBV which encodes gp350/220) that had been digested withXbaI and BamHI and blunt-ended. The resulting plasmid, pRB4298, has theEBV gp350/220 coding sequences at -220 relative to the transcriptionalstart site of the synthetic promoter. pRB4298 was partially sequenced atthe 5' end of the α4 portion of the synthetic promoter to verifyorientation.

To provide flanking homologous sequences to allow recombination of thesynthetic α4-γ₁ U_(L) 19 promoter/gp350/220 DNA construct into thegenome of R7020, a plasmid was constructed as a shuttle vector whichcontained a genomic deletion identical to the deletion in the naturalvital thymidine kinase gene of R7020 and which had a suitable polylinkerfor cloning other genetic elements. pRB173 (Post et al., supra) whichcontains the BamHI Q segment of HSV(F) in pUC9 was digested with BglIIand SacI to delete 500 bp of the sequences of U_(L) 23 and U_(L) 24, theidentical deletion present in R7020. Into the plasmid was introduced adouble stranded synthetic oligonucleotide having the following topstrand sequence: ##STR2## The introduction of the oligonucleotiderestored the BglII and SacI restriction sites, induced restriction sitesfor KpnI and XbaI (which are not otherwise present in the pUC9 vector)and produced bidirectional polyadenylation signals. The resultingshuttle vector was designated pRB3982.

A 3000 bp fragment of pRB4298 containing the synthetic α4-γ₁ U_(L) 19promoter/gp350/220 DNA construct was obtained by digestion with KpnI andHincII. The fragment was ligated into the shuttle vector pRB3982 whichhad been digested with XbaI, treated with T4 polymerase anddeoxynucleotides, and then cut with KpnI. The resulting plasmid,pRB4299, contains the natural polyadenylation and transcriptionaltermination signals of the gp350/220 gene at the 3' terminus of thesynthetic α-γ promoter/gp350/220 DNA construct. The synthetic promoterlies toward the truncated U_(L) 24 gene. FIG. 6 is a schematicrepresentation of the construction wherein NarI, BamHI, BstVI denoterestriction enzyme recognition sites; TATA denotes the criticaltranscriptional initiation signal; boxed regions indicate transcribed,nontranslated portions of the various promoters; ATG denotes themethionine codon at which translation is initiated; numbers indicatepositions relative to the transcriptional initiation site of the nativepromoter; and boxed regions below the lines indicated regions which wereverified by sequencing. The first line of FIG. 6 displays the featuresof the natural promoter of the α4 gene from pRB168. The γ1 ICP5 promoterin the second line is the natural promoter of the γU_(L) 19 gene frompRB140. The third line indicates the arrangement of the syntheticpromoter. The fourth line sets out the arrangement of the syntheticpromoter attached to the gp350/220 gene flanked by homologous sequencesderived from pRB3982 as present in pRB4299.

To obtain recombinant virus R7125 (ATCC VR 2389), vital DNA of R7020 andplasmid DNA of pRB4299 were cotransfected into rabbit skin cells by themethod described in Example 1. Plaques were selected for expression ofgp350/220 by treatment with anti-gp350/220 antibody followed by stainingwith immunoperoxidase. Complement free media was used after it wasdetermined that the staining reaction resulted in complement activationand destruction of the recombinant virus infected cells. After 5 roundsof serial selection, R7125 was determined to be free of contaminatingR7020 parent virus.

EXAMPLE 4

Recombinant virus R7125 was serially passaged in Vero cells seven times,and the passaged virus was analyzed with regard to its DNA restrictionprofile. The expression of gp350/220 by nonpassaged R7125 and passagedR7125 (R7125P7) in comparison to parent virus R7020 was also analyzed.

Stability of R7125 after serial passage was assessed by DNAhybridization. Viral DNA was digested with EcoRI or EcoRV, separated in8% agarose in Tris-phosphate at 70 volts overnight, stained withethidium bromide and photographed. DNA was then transferred by gravityand capillary action to nitrocellulose and hybridized with gp350/220 DNAwhich had been nick translated and radiolabelled with ³² P-dCTP.Resulting bands are shown in FIGS. 7A (EcoRI) and 7B (EcoRV), whereinbands (denoted by the number 1) representing the added HSV-2 genes("added" in comparison to R3410) in R7020 are demonstrated to be presentin R7125 and unique bands in R7125 (denoted by the number 2) contain the˜3000bp synthetic α4-γ₁ U_(L) 19 promoter/gp350/220 insert whichcontains an EcoRI but not an EcoRV recognition site. The unique bandsintroduced by the added sequences in R7125 are stable in R7125.

Recombinant viruses R7020 and R7125 were compared for expression of EBVgp350/220. Extracts of Vero cells (˜4×10⁵ cells) which were infectedwith 5 pfu virus were harvested at 2, 4, 6 and 14 hours post-infectionafter labelling with S³⁵ -Met for the last hour before harvest. Extractswere electrophoresed in 5% bis-acrylamide gels then transferred tonitrocellulose and gp350/220 was detected by anti-gp350/220 monoclonalantibody followed by alkaline phosphatase conjugated goat anti-mouseantibody. Results of the assay are presented in FIG. 8. While R7020extracts did not contain detectable gp350/220, expression of gp350/220increased over time in R7125 extracts. The upper bands in theantibody-treated R7125 lanes of FIG. 8 represent authentic gp350 andspliced gp220 as occurs in EBV-infected lymphocytes, while the lowerbands represent precursors to the glycosylated mature protein. Extractsof cells infected with a R7125P7 isolate contained similar quantities ofgp350/220 when compared to extracts of cells infected with nonpassagedR7125. Kinetics of expression of gp350/220 by nonpassaged and passagedR7125 appear to be similar with large quantities appearing between 2 and4 hours and continuing to 14 hours.

The foregoing illustrative examples relate to presently preferredembodiments of the invention and numerous modifications and variationsthereof will be expected to occur to those in the art. Thus only suchlimitations as appear in the appended claims should be placed upon thescope of the present invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 82 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCCTGGGGGGCGGCGGGGGGCCGGCGGCCTCCGCTGCTCCTCCTTCCCGGCGGCCGCTGG60                GACTATATGAGCCCGAGGATCC82                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 797 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGCCAGTGCTCGCACTTCGCCCTAATAATATATATATATTGGGACGAAGTGCGAACGCTT60                CGCGTTCTCACTTCTTTTACCCGGCGGCCCCGCCCCCTTGGGGCGGTCCCGCCCGCCGGC120               CAATGGGGGGGCGGCAAGGCGGGCGGCCCTTGGGCCGCCCGCCGTCCCGTTGGTCCCGGC180               GTCCGGCGGGCGGGACCGGGGGGCCCGGGGACGGCCAACGGGCGCGCGGGGCTCGTATCT240               CATTACCGCCGAACCGGGAAGTCGGGGCCCGGGCCCCGCCCCCGGCCCGTTCCTCGTTAG300               CATGCGGAACGGAAGCGGAAACCACCGGATCGGGCGGTAATGAGATGCCATGCGGGGCGG360               GGCGCGGGCCCACCCGCCCTCGCGCCCCGCCCATGGCAGATGGCGCGGATGGGCGGGCCC420               GGGGGTTCGACCAACGGGCCGCGGCCACGGGCCCCCGGCGTGCCGGCGTCGGGGCGGGGT480               CGTGCATAATGGAATTCCGTTCGGGGCGGGCCCGCCTGGGGGGCGGCGGGGGGCCGGCGG540               CCTCCGCTGCTCCTCCTTCCCGGCGGCCGCTGGAACTATATGAGCCCGAGGATCCCACGT600               CCCCGGGGTCTGTTGGGGACACTGGGTTCTCCTGGAACGAGGCCGCAGCCTTCTCCCGGT660               GCCTTTCCCCCCCGACCGACACCCGGCCTCTCACACAGCATCCCCCGCCTCTTTGGGTCC720               GGGTCCGTCGTGTCGTCTTTCGGTGGACCTTGGGCCGTCGGGCACGTACACGGGTGGCCG780               GGCGTTGGGGTGGATCC797                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATCTTTATTAGGTACCCTCTAGAATAAAGAGCTAAATAATCCATGGGAGATCTTATTTC60                __________________________________________________________________________

What is claimed is:
 1. A method of inducing antibody production againsta polypeptide from a disease-causing organism in a host, said methodcomprising the step of administering an antibody-inducing dose of arecombinant herpes simplex virus to said host, said recombinant herpessimplex virus comprising a recombinant herpes simplex virus genomecomprising a synthetic herpes simplex virus promoter and a heterologousgene operatively linked 3' to said synthetic promoter, said syntheticpromoter comprising a herpes simplex virus α gene promoter fragmentoperatively linked 5' to a herpes simplex virus γ gene promoterfragment, wherein said polypeptide is encoded by said heterologous gene,said heterologous gene being derived from said disease-causing organism.2. The method of claim 1 wherein said herpes simplex virus α genepromoter fragment comprises promoter sequences upstream of thetranscription initiation site of said α gene.
 3. The method of claim 1wherein said herpes simplex γ gene promoter fragment comprises thetranscription initiation site and the transcribed non-coding sequencesdownstream of the transcription initiation site of said γ gene.
 4. Themethod of claim 1 wherein said herpes simplex virus α gene promoterfragment comprises promoter sequences upstream of the transcriptioninitiation site of the α4 gene and said herpes simplex virus γ genepromoter fragment comprises the transcription initiation site and the 5'transcribed non-coding sequences of the γ₁ U_(L) 19 gene.
 5. The methodof claim 1 wherein said heterologous gene encodes part or all of theEpstein-Barr virus glycoprotein gp 350/220.
 6. The method of claim 1wherein the recombinant herpes simplex virus is the virus deposited asATCC VR
 2389. 7. The method of claim 2 wherein said herpes simplex virusα gene promoter fragment is an α4 gene promoter fragment.
 8. The methodof claim 7 wherein said herpes α4 gene promoter fragment comprisesnucleotides -588 to -12 of said α4 gene promoter.
 9. The method of claim3 wherein said herpes simplex virus γ gene promoter fragment is a γ₁U_(L) 19 gene fragment.
 10. The method of claim 9 wherein said herpessimplex virus γ₁ U_(L) 19 gene fragment comprises bases -11 to +189 ofsaid γ₁ U_(L) 19 gene promoter.